Stem cells are primitive cells which are capable of self-renewal, and ultimately become differentiated into specific cell types of defined function. Stem cells are capable of proliferation either to generate further identical stem cells, or to produce more differentiated cell types. Later more differentiated cell types always become more differentiated on proliferation. Stem cells exist for most tissue types but are continuously active in the skin and mucosal systems, and for the blood and bone marrow (haematopoietic stem cells).
Haematopoietic stem cells are capable of self-renewal, multilineage proliferation and differentiation, and long-term support of the haematopoietic and lymphoid systems. They form a subpopulation within the Haematopoietic progenitor compartment (HPC), which mainly comprises cells of more limited potentiality. HPC cells are mainly located within the bone marrow stroma, where complex interaction with stromal cells, extracellular matrix components and cytokines, permits regulation of cell proliferation and differentiation. HPC cells are also present in the blood under a variety of physiological, pathological and iatrogenic circumstances. HPC can be harvested from bone marrow or peripheral blood, and will re-engraft the bone marrow following intravenous infusion in patients who have received ablative (i.e. destructive) doses of chemotherapy and/or radiotherapy, leading to regeneration of haematopoiesis and immunity. Thus, HPC cell transplantation is of considerable clinical utility in the management of patients with haematological and solid malignancies, bone marrow failure, and inborn errors of haematopoiesis, immunity or metabolism.
There is thus a need for supplies of autologous HPC cells which may be cultured in vitro prior to reintroduction into a patient whose HPC cell population has been depleted due to chemotherapy and/or radiotherapy. The populations of such HPC cells may take many weeks or months to recover naturally to their normal levels. The use of autologous cells from the patient himself avoids rejection of the transplanted cells. An object of the present invention is to address the culturing of such HPC cells in a convenient manner. This is carried out with a view to improving the rate and durability of haematopoietic recovery, the removal of any neoplastic contamination, and the possible use of HPC calls as a vehicle for immunotherapy and gene therapy.
In vivo, HPC cells are generally located within the bone marrow stroma. In vitro, HPC cells are able to adhere to bone marrow stromal layers before proliferating and releasing more committed progenitors. Stem cells undergo marked proliferation and differention into multiple lineages, ultimately giving rise to fully differentiated cells, such as red blood cells, platelets, a variety of white blood cells, and also immune cells such as T lymphocytes and B lymphocytes. Thus, the reintroduction of HPC cells or stem cells into the patient who is depleted therein, allows efficient repopulation of these haematopoietic cell types.
Although stromal layers may provide a suitable substrate for HPC cell immobilization and culture, such stromal layers have a number of disadvantages. Firstly they are fragile. This imposes limitations on the types of systems which may be employed to culture such cells. The growth of HPC cells in vitro requires a rigid substrate on which the layers of stromal cells can be grown in order to maintain the integrity of the stroma. Moreover, it is found that HPC cells grown on stroma in this way only have a limited storage lifetime, of about six to eight weeks due to death of the stromal cells. A further problem is that the use of the stromal cells for the growth of HPC cells is limited to HPC cells derived from bone marrow, and cannot be used to culture other HPC cell types, such as mucosal cells.
In the case of an autologous graft, there may be a need to culture HPC cells for re-engrafting using stromal cells which are also derived from the patient himself. Immunological problems of rejection may arise if non-autologous stromal cells are used to culture the stem cells. The need to first collect and then grow a layer of the patient's stromal cells before they can be used to culture his HPC cells adds to the time and complexity of the production of the autologous HPC cells. In the case of bone marrow, a sample thereof contains not only stem cells but also the required stromal cells. However, if stem cells are harvested from peripheral blood or cord blood stromal cells are absent and have to be provided from a non-autologous source, leading to possible immunological problems.
Finally, the stromal cultures contain an ill-defined set of cells, growth factors etc. which renders the controlled culturing thereof very difficult if reproducible stromal cultures or predictable characteristics are to be obtained.
It is an object of the present invention to mitigate these problems.